1. Field of the Invention
The present invention relates to a process for purification of xanthophylls from plant extracts, particularly marigold oleoresins.
2. Description of the Related Art
The carotenoids comprise a group of natural pigments found abundantly in plants, some fish, crustaceans, birds, algae and bacteria. Within this group of pigments are the yellow carotenoids, including both the carotenes (e.g., β-carotene) and xanthophylls (e.g., lutein and zeaxanthin), and the red carotenoids, including capsanthin, canthaxanthin and astaxanthin. These yellow and red carotenoids are often present in plants, especially flowering plants, together with other classes of pigments, including primarily green chlorophyll pigments.
Carotenoids and in particular, xanthophylls, from marigold extracts have been used for decades in the poultry industry for pigmentation of broiler skins and egg yolks. Lutein, is present at much higher concentrations than zeaxanthin in marigold extracts. Pigmenting formulations for use in the poultry industry having relatively high concentrations of zeaxanthin are known, wherein the lutein has been isomerized to yield zeaxanthin (U.S. Pat. No. 5,523,494 to Torres and U.S. Pat. No. 5,973,211 to Rodriguez).
In addition to their commercial importance in the poultry industry, the carotenoids have recently received considerable attention from scientists with respect to their potential role in promoting human health. Compounds like α and β-carotene, lutein and zeaxanthin have been shown to possess strong antioxidant activity, which may retard or prevent diseases like cancer, arteriosclerosis, cataracts, macular degeneration and others (Bowen, WO98/45241). Lutein and zeaxanthin are the only carotenoids present in the macular region of the human retina and are related to the normal function of the macula responsible for visual acuity. It has also been reported that carotenoids enhance the immune response. Free radicals produced as byproducts of metabolic processes and originating from environmental pollutants (such as nitrogen dioxide and ozone of polluted air, heavy metals, halogenated hydrocarbons, ionizing radiation and cigarette smoke) are implicated as causative factors in many of the above-mentioned diseases. Carotenoids are potent quenchers of the highly reactive oxygen free radicals that can initiate a cascade of detrimental chemical reactions. Carotenoids also function as chain-breaking antioxidants, especially at low partial pressures of oxygen. Thus, carotenoids can work to quench free radical-induced reactions and can also prevent generation of free radicals, thereby limiting free radical/oxidative damage.
Khachik has described (U.S. Pat. No. 5,382,714) a process for obtaining lutein, the purity of which is usually greater than 90%, determined by UV/visible spectro-photometry. The purity of the lutein was found to be around 94.79%, its isomers around 3.03% based on HPLC analysis and others consisting of zeaxanthin, etc. The main drawback of the above mentioned process is in the use of a halogenated solvent. Halogenated solvents are banned for use in human food applications in most of the countries because of apprehensions about their potential carcinogenic effects.
Ausich and Sanders (U.S. Pat. No. 5,648,564) have developed a process for obtaining xanthophyll crystals containing approximately 70-85% total carotenoids, deemed to contain substantially pure xanthophylls. The HPLC analysis of the xanthophylls showed 85-95% trans-lutein, 0.2-1.5% of its geometrical isomers, 2.5-8% zeaxanthin. The poor solubility of xanthophyll esters in propylene glycol and the subsequent heating to temperatures around 70° C. for 10 hours are the main disadvantages of the above mentioned process, since the lutein undergoes isomerization and decomposition under the above conditions. Further, propylene glycol is not a cost- affordable solvent from commercial considerations.
Khachik in his U.S. Pat. No. 6,262,284 has developed a process for obtaining lutein and zeaxanthin crystals (97% pure) starting from marigold meal instead of marigold extract. This process involved simultaneous extraction and saponification of xanthophyll esters. The main limitations in the above process are saponification of the extract without concentration leading to consumption of large volumes of solvents that are difficult to manage in commercial production. Further formation of peroxides from solvents like THF may cause degradation of the xanthophylls. Also, the use of silica-gel column chromatography is a cumbersome and less economic process for commercial scale production of pure lutein crystals.
Madhavi and Kagan (U.S. Pat. No. 6,380,442) have reported a process for the isolation of mixed carotenoids from plants and illustrated the same with examples of marigold oleoresin. The hydrolysis temperature is high and the reaction time is long leading to lutein oxidation and degradation. The method is not attractive for commercial applications since the water required is more than 30 times per kg of the input material and the lutein is released as minute crystals dispersed in a slimy soapy solution making recovery difficult.
Rodriguez et al. (U.S. Pat. No. 6,329,557) have disclosed an industrial scale process for obtaining xanthophyll crystals from marigold extract. The method is useful for marigold oleoresins and provides an industrial scale process for obtaining lutein and zeaxanthin concentrates of high purity using saponified marigold extracts.
Montoya, et al (U.S. Pat. No. 6,504,067) describe a process for cleaning oleoresin with alkali and acid. The cleaned oleoresin is subjected to aqueous alkali hydrolysis at a temperature of 90° C. for 8 hours in the presence of emulsifiers. This method suffers the disadvantage that high temperature and long cycle time leads to degradation of free lutein.
Kumar, et al. (U.S. Pat. No. 6,743,953) disclose a method which includes saponification in an alcohol to avoid addition of water, removing the alcohol under reduced pressure, and extraction of xanthophylls in ethyl acetate to obtain xanthophylls in good yield. The method of Kumar, et al. avoids the use of water during saponification so that steps to remove the water using organic solvents, particularly halogenated organic solvents, can be avoided. Saponification in alcohol was known as taught by Grant (U.S. Pat. No. 3,523,138).
Sadano, et al. (U.S. 2004/0055954) discloses extraction of marigold oleoresin with supercritical fluid extraction such as high pressure carbon dioxide. Selective extraction is achieved by changing the pressure of the supercritical fluid. The extracted oleoresin is further purified by dissolving in a ketone solvent and removing the precipitate. The requirement for special equipment for supercritical fluid extraction makes this method unattractive for commercial applications.
Khachik (U.S. Application No. 2005/0038271) disclose a method of extracting zeaxanthin from Lycium Chinese Mill berries and lutein from marigold without using harmful organic solvents. The method employs tetrahydrofuran and an FDA Class 3 alcohol, preferably ethanol, as the extraction solvents. However, as mentioned above, formation of peroxides from solvents like THF may cause degradation of the xanthophylls.
Quesnel (U.S. Application No. 2005/0139145) discloses a relatively simple method of purifying carotenoids which include lutein using organic solvents. The crystals obtained by this procedure were high in all trans lutein.
Rosales, et al. (U.S. Application No. 2005/0153002) disclose a process to obtain xanthophylls in good yield and high purity that are free from epoxized derivatives. The method includes drying marigold flowers under mild conditions to avoid formation of epoxides, followed by saponification and use of metallic halogenides (e.g. calcium chloride) to remover fatty acids. The resulting precipitate is removed and washed with polar solvent (eg. Alcohol, acetone).
Bhaskaran, et al. (U.S. Application No. 2005/0182280) disclose a method of preparing a stable lutein paste from oleoresin using relatively low temperatures and short reaction times. However, the method includes multiple steps such as dissolving in alcohol, cleaning on an ion exchange resin, hydrolysis of the esters using a phase-transfer catalyst, quenching with an acidic solution, dissolving and filtering the solids, drying the esters, triturating the residue, and distilling the alcoholic fraction. Accordingly, the described method is not a commercially viable method in view of the large number of steps involved.
Clearly there remains a need for a simple, adaptable method for isolation of free xanthophylls that limits the use of organic solvents and minimizes degradation and epoxide levels in the xanthophyll product. Preferred embodiments of the present invention are directed to an improved process for the isolation of xanthophylls, which are free of epoxide derivatives, from plant extracts which is adaptable, economic and easy to carry out.